Micro-Scale Engineering III Lab-on-a-Chip for Sorting Cells. Y. C. Lee

Transcription

1 Micro-Scale Engineering III Lab-on-a-Chip for Sorting Cells Y. C. Lee Department of Mechanical Engineering University of Colorado Boulder, CO Guest lecture on Thursday: Ramsey Zeitoun; Biofuel February 4,

2 High-throughput microfluidic single-cell RT-qPCR A. K. White et al., "High-throughput microfluidic single-cell RT-qPCR," PNAS, 2011 Aug 23; 108(34):

3 An Integrated Nanoliter DNA Analysis Device (Mark Burns et al., Science, 1998) 47mm X 5mm X 1mm Reagent solution Bso B1 restriction enzymes Bst DNA polymerase enzymes Intercalatng dye Electronic signals DNA solution DNA fragments amplification site-specific oligonucleotides Outside sources: Pressure Electronic control Optical excitation source 3

4 Homework #2 Write a one-page report for the comparison by reporting: 1) summary of comparison 2) cell capture and lysis 3) reverse transcription 4) PCR and real-time monitoring 5) number of parallel processing units 6) another feature of your interest The purpose of this comparison is for you to appreciate the significant advancement in the lab-on-a-chip technology achieved during the last 15 years. Hope your independent project will identify a device that may make an impact 15 years from now. 4

5 Single cell digital PCR device White et al., Anal. Chem., 2013, 85 (15), pp

6 Inertial focusing for tumor antigen dependent and independent sorting of rare circulating tumor cells E. Ozkumur et al., Sci. Transl. Med. 5, 179ra47 (2013). 6

7 Antibodies latched onto the protein EpCAM 7

8 Labelling Efficiency For positive selection, pre-stained cell lines with low and high EpCAM expression were spiked into whole blood, incubated with various amounts of anti-epcam coated beads, and samples were mixed using either active magnetic mixing or passive mixing. Following mixing, samples were processed through a debulking array to remove RBCs and collected in a 24-well plate; target cells were identified 8 based on their fluorescence and their bead loading was evaluated.

9 Complete System 9

10 Inertial focusing for tumor antigen dependent and independent sorting of rare circulating tumor cells (CTCs) Positive depletion identifies CTCs using antibodies that latch onto the protein EpCAM, commonly found on the surface of CTCs. Current commercially available CTC-sorting devices are based on positive depletion, and the CTC-iChip also successfully isolated magnetically labeled CTCs with enhanced performance. E. Ozkumur et al., Sci. Transl. Med. 5, 179ra47 (2013). 10

11 Laminar and Turbulent Flows 11

12 Laminar Flow in a Pipe Re= u m D/µ =1, kg/m³ u m = m/sec µ = (N s)/m 2 D=100E-6 m Re= 5 D=1,000E-6 m Re=50 12

13 Flow Past a Sphere 13

14 Flow Past a sphere Re <1 1<Re< <Re< 2E5 Re= u m D/µ =1, kg/m³ µ = (N s)/m 2 D=10E-6 m (particle) u m = m/sec Re= 0.05 u m = m/sec Re=

15 Continuous Particle Separation Through Deterministic Lateral Displacement Lotien Richard Huang et al., Science 304, 987 (2004). (A) Geometric parameters defining the obstacle matrix. A fluid flow is applied in the vertical direction (orange arrow). (B) Three fluid streams (red, yellow, and blue) in a gap do not mix as they flow through the matrix. Lane 1 at the first obstacle row becomes lane 3 at the second row, lane 3 becomes lane 2 at the third row, and so on. Small particles following streamlines will thus stay in the same lane. (C) A particle with a radius that is larger than lane 1 follows a streamline passing through the particle's center (black dot), moving toward lane 1. The particle is physically displaced as it enters the next gap. Black dotted lines mark the lanes. 15

16 Continuous Particle Separation Through Deterministic Lateral Displacement 16

17 Inertial focusing for tumor antigen dependent and independent sorting of rare circulating tumor cells E. Ozkumur et al., Sci. Transl. Med. 5, 179ra47 (2013). 17

18 Hydrodynamic size based separation A single debulking array has 24 parallel channels and arrays are 150 μm in depth. Each of the 24 parallel channels has two input streams that run side by side in laminar flow. (A) Building on previously established design principles, we developed two different array configurations, Array 1 and Array 2. The gaps between posts (g) were 20 and 32 μm for Arrays 1 and 2, respectively. The center to center distances between the posts (λ) were 35 and 56 μm, respectively, and the row shift fraction (ε) was 0.16 for both array designs. (B) Cell retention and debulking efficiencies were characterized for both of the arrays in multiple experiments (n > 10). (C) In our current study, we measured CTC diameters between 8 and 20 μm (Fig. 6). Therefore, we estimated a lower size cut off of 8 μm, and larger size range going up to 30 μm. We concluded that the approximated CTC size range 18 overlaps best with Array 2.

19 Continuous inertial focusing, ordering, and separation of particles in microchannels Dino Di Carlo et al., Continuous inertial focusing, ordering, and separation of particles in microchannels, , PNAS,

20 Inertial Focusing 20

21 Inertial focusing for tumor antigen dependent and independent sorting of rare circulating tumor cells WBC CTC E. Ozkumur et al., Sci. Transl. Med. 5, 179ra47 (2013). pos CTC-iChip 21

22 Magnetic Deflection Undeflected Deflected 22

23 neg CTC-iChip Negative Depletion Negative depletion, in contrast, sorts out CTCs by eliminating all other known cells first. By magnetically labeling white blood cells rather than CTCs, the researchers were able to isolate a vast array of unlabeled tumor cells using CTC-iChip. Negative depletion allows for the detection of CTCs without having to know what type of tumor they came from beforehand and regardless of whether they produce EpCAM. Thus, negative depletion methods may be able to identify a greater variety of tumors across a broader range of development than positive depletion. 23

24 Inertial focusing for tumor antigen dependent and independent sorting of rare circulating tumor cells CTC WBC E. Ozkumur et al., Sci. Transl. Med. 5, 179ra47 (2013). neg CTC-iChip 24

25 Overall system performance using cancer cell lines spiked into whole blood. SKBR3 human breast cancer cells Human prostate PC3-9 cancer cells MDA-MB-231, triple-negative mesenchymal breast cancer MCF10A breast cancer LBX1: Epithelial-mesenchymal transition (EMT) master regulator 25

26 CTC isolation by pos CTC-iChip in cancer patients The sensitivity of the CTC-iChip is particularly critical in patients with a lower CTC burden. 26

27 CTCs with cytopathology and ICC stain Immunocytochemistry (ICC) 27

28 Variation of CTC sizes and morphologies 28

29 Heterogeneity of RNA expression between CTCs isolated from a prostate cancer patient (qrt-pcr) 29

30 Inertial focusing for tumor antigen dependent and independent sorting of rare circulating tumor cells CTC WBC E. Ozkumur et al., Sci. Transl. Med. 5, 179ra47 (2013). neg CTC-iChip 30

31 Inertial focusing for tumor antigen dependent and independent sorting of rare circulating tumor cells The CTC-iChip was able to sort CTCs from whole blood: - quicker than previously developed microfluidic devices, - allowing larger blood samples to be processed in a short amount of time more efficiently than other magnet-based sorting systems, - reducing the amount of materials required and increasing the sensitivity of the device - more effectively in samples with few EpCAM-producing CTCs compared to other sorting methods - more effectively in samples known to not express EpCAM, such as triple negative breast cancer and melanoma. By collecting CTCs in a way that allows them to be studied further, the CTC-iChip could also help clinicians identify important genetic differences between individual CTCs that may inform which targeted therapy is indicated. It will enable, in the long run, [a physician] to treat the right patient with the right drug at the right dose at the right time. 31